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Abstract

Background

Brief heat stimuli that excite nociceptors innervated by finely myelinated (Aδ) fibers
evoke an initial, sharp, well-localized pain ("first pain") that is distinguishable
from the delayed, less intense, more prolonged dull pain attributed to nociceptors
innervated by unmyelinated (C) fibers ("second pain"). In the present study, we address
the question of whether a brief, noxious heat stimulus that excites cutaneous Aδ fibers
activates a distinct set of forebrain structures preferentially in addition to those
with similar responses to converging input from C fibers. Heat stimuli at two temperatures
were applied to the dorsum of the left hand of healthy volunteers in a functional
brain imaging (fMRI) paradigm and responses analyzed in a set of volumes of interest
(VOI).

Results

Brief 41°C stimuli were painless and evoked only C fiber responses, but 51°C stimuli
were at pain threshold and preferentially evoked Aδ fiber responses. Most VOI responded
to both intensities of stimulation. However, within volumes of interest, a contrast
analysis and comparison of BOLD response latencies showed that the bilateral anterior
insulae, the contralateral hippocampus, and the ipsilateral posterior insula were
preferentially activated by painful heat stimulation that excited Aδ fibers.

Conclusions

These findings show that two sets of forebrain structures mediate the initial sharp
pain evoked by brief cutaneous heat stimulation: those responding preferentially to
the brief stimulation of Aδ heat nociceptors and those with similar responses to converging
inputs from the painless stimulation of C fibers. Our results suggest a unique and
specific physiological basis, at the forebrain level, for the "first pain" sensation
that has long been attributed to Aδ fiber stimulation and support the concept that
both specific and convergent mechanisms act concurrently to mediate pain.

Background

There is substantial evidence that pain is mediated by two classes of nociceptive
afferent fibers, finely myelinated Aδ fibers and unmyelinated C fibers [1,2]. Following a brief (< 1 sec) noxious cutaneous heat stimulus, two distinct sensations
arise: an initial, sharp pain thought to be mediated by Aδ fibers ("first pain") and
a delayed, less intense, more prolonged, heat sensation ("second pain") that is attributed
to the excitation of C fibers [2,3]. These two pain experiences have unique psychophysiological and pharmacological characteristics
[3-5], supporting a long-standing hypothesis that each fiber type activates central pathways
that are anatomically unique although partially overlapping [2]. Functional imaging studies show that a number of brain regions are active during
pain including the primary and secondary somatosensory cortex, insula, cingulate cortex
and thalamus [6-8], but the relative contribution of each fiber type to the elicited BOLD responses
are unknown. Electrophysiological studies, using magnetoencephalography, evoked potentials
(EP), and selective laser stimulation of Aδ and C fibers show very similar locations
of the early cerebral activations from these sources [7,9-11]. Qiu and colleagues used laser stimulation and functional magnetic resonance imaging
(fMRI) to show that the cerebral activations following preferential Aδ fiber stimulation
overlap those evoked by preferential C fiber stimulation although the C activations
were significantly greater in the bilateral anterior insulae and the ipsilateral medial
frontal gyrus [12]. The results reported by Qiu and colleagues [12] suggest an extensive anatomical overlap of structures activated by painful Aδ and
C fiber stimulation; however, an anatomically unique aspect of Aδ-mediated pain remains
in question. A related functional imaging study by Veldhuijzen and colleagues also
revealed an extensive overlap of structures activated during equally intense sharp
or burning pain following diode laser stimulation at parameters designed to favor
Aδ or C fiber excitation, respectively [13]; however, some temporal and parietal lobe structures were more active during sharp
pain while the dorsolateral prefrontal cortex responded more during burning pain.
There is also uncertainty about whether the unique cerebral responses to the brief
excitation of C fibers are related specifically to pain. This question arises because
brief cutaneous heat stimulation above C, but below Aδ fiber threshold, is not reliably
painful [12,14,15] and because cerebral structures activated by C fibers responding to warm or tactile
stimuli [16] may also respond during Aδ-mediated pain.

In the present study, the aim was to identify the cerebral mechanisms that could be
uniquely involved in mediating the brief, sharp "first pain" that is mediated by Aδ
fibers. This was done by comparing responses to a noxious heat stimulus that excites
cutaneous Aδ fibers with responses to converging input from C fibers. We used brain
evoked potentials to identify the brief heat stimuli that preferentially excite Aδ
and C fibers and we used functional magnetic resonance imaging (fMRI) to compare quantitatively
the brain responses to each of these stimuli. We used contrast imaging and temporal
analysis to detect the preferential activation of brain structures by Aδ fibers.

We found that two sets of forebrain structures mediate the initial sharp pain evoked
by brief cutaneous heat stimulation. One set responding preferentially to the brief
stimulation of Aδ heat nociceptors and those with similar responses to converging
inputs from the stimulation of C fibers.

Methods

Subjects

Seven right handed volunteers (age 18-36 years old; three females) participated in
the experiment. All subjects were free of medication and had not consumed caffeine
or other psychoactive drugs on the day of testing. All subjects gave informed consent
before testing. The experimental protocol was conducted in accordance with the Helsinki
declaration and was approved by the Internal Review Boards of the Veterans Affairs
Medical Center (Ann Arbor) and the University of Michigan, Ann Arbor, MI, USA.

Somatosensory stimuli

We used a contact-heat evoked potential stimulator (CHEP stimulator; Medoc, Ramat Yishai, Israel). The thermode was strapped to the hand
and not moved between stimuli. The thermode contacted a cutaneous area of 573 mm2 and is comprised of a heating thermofoil (Minco Products, Inc., Minneapolis, Minnesota)
covered with a 25 micron layer of thermoconductive plastic (Kapton®). Two thermocouples embedded 10 microns within this conductive coating provide an
estimate of the skin temperature at the thermode surface. The thermofoil-skin interface
temperature is measured and software-controlled 150 times per second. Heat stimuli
were delivered at intensities of 41°C and 51°C. From a contact baseline temperature
(35°C), the average time from onset to peak temperature is on average 190 ± 24 ms
for 41°C and 250 ± 8 ms for 51°C. The full-width-half-maximum (FWHM) of the heat pulse
is 350 ms. As reported previously, the stimulus parameters selected here reliably
evoke quantifiable contact heat evoked potentials (CHEPs) mediated through C fibers
accompanied by a warm sensation (41°C) or mediated through Aδ fibers accompanied by
a sharp, pricking pain (51°C) [14,17]. With stimuli stronger than 41°C, an earlier potential associated with Aδ fiber excitation
may begin to appear and the sensation is more like to be slightly painful (see Results).
With stimuli less than 51°C, the positive potential associated with Aδ fiber stimulation
and the sensation of pricking pain are less reliably present. Stimulation above this
intensity is often associated with a delayed burning sensation suggesting the excitation
of C fibers. Therefore, the stimulus parameters we used here for brief heat pulses
were selected to emphasize the electrophysiological and psychophysical characteristics
of "first pain" and maximize the combined electrophysiological and psychophysical
differences between Aδ and C fiber excitation.

Experimental protocols

Evoked potentials (EPs) and functional magnetic resonance images (fMRI) were acquired
in two sessions one to four weeks apart. All subjects participated in both sessions
and received up to 200 stimuli in each session; 20 runs of 10 stimuli divided into
4 runs of five different modalities (epidermal electrical, 41°C to hairy and glabrous
skin, and 51°C to hairy and glabrous skin) (see Figure 1). Most subjects received fewer than 200 stimuli during the EP session, because two
runs of each stimulus modality were usually enough to provide an easily identifiable
brain potential. Responses to electrical stimuli and to heat stimuli delivered to
glabrous skin will be reported elsewhere; hence the present article reports responses
only to 41°C and 51°C heat stimuli delivered to hairy skin on the dorsum of the left
hand. The inter-stimulus-interval (ISI) varied randomly between 11 and 15 s. The stimulus
electrode/thermode was not moved between stimuli.

Figure 1.Experimental protocol of fMRI experiment. The top row show the messages displayed to the subject in the scanner for each somatosensory
stimulus. The stimulus was delivered after a 2-6 s delay ("Ready" message), followed
by a 5 s "Wait" message before a 4 s "Rate stimulus" message was displayed, at which
time the subject rated the stimulus on a five-button claw. The middle row illustrates
how one stimulus (bars) is repeated 10 times, creating one run. The bottom row illustrates
how each run is repeated with different stimulus modalities, making a total of 20
runs. Only stimulus intensities delivered to hairy skin (41°C and 51°C) is reported
here. Intermingled were two heat stimulus intensities delivered to glabrous skin and
one epidermal electrical stimulus. Results from these stimuli are reported elsewhere.

Intensity ratings

The perceived intensity was rated after each stimulus, according to a 5-point numerical
rating scale (NRS): 1 - 'not perceived', 2 - 'perceived but not painful', 3 - 'barely
painful', 4 - 'moderately painful' and 5 - 'very painful'. Before data acquisition,
the subject was instructed to pay close attention to the stimulus site and to rate
the intensity of each stimulus approximately two seconds after it was perceived (EP)
or when a visual cue appeared (fMRI). The ratings were performed verbally (EP) or
via a five-button claw attached the right hand (fMRI). The five-button claw was a
custom designed key pad strapped to the hand having one button for each finger (thumb
= 1, little finger = 5). Pain intensity ratings and EP latencies were not normally
distributed and therefore compared with the non-parametric Wilcoxon test. The Mann-Whitney
U test was used for post-hoc comparisons; p < 0.05 was considered statistically significant.
These statistical measures were carried out using SPSS (SPSS 15; SPSS Inc., Chicago,
Illinois, USA).

EP acquisition and analysis

Before recordings, the subjects were presented with the different stimuli and practiced
subjective ratings. Recordings were made from the vertex (Cz), referenced to bilateral
earlobes (A1+A2), using a standard EEG cap and Neuroscan software (Scan 4.2, Compumedics,
El Paso, Texas). The electrooculogram (EOG) was recorded from supra- and infraorbital
electrodes for offline artifact rejection. The impedance was maintained below 5 kΩ.
The signals were amplified 100 000 times, sampled at 500 Hz, bandpass filtered at
0.1 - 30 Hz and notch filtered at 60 Hz. Subjects were seated in a padded reclining
chair, room temperature was 22-23°C, and skin temperature was always above 30°C. The
subjects were instructed to keep their eyes open, to focus on a fixed point at the
wall and to avoid blinking. During offline analysis, the continuous EEG signal was
split into epochs relative to stimulus onset using EEGLAB 6 [18]. Each epoch was visualized and discarded if contaminated by ocular artifacts. The
remaining sweeps were averaged for each stimulus modality and subject. The peak latency
of the major negative and positive component was identified by visual inspection of
the averaged response. Peak latencies were compared with Wilcoxon paired non-parametric
test (SPSS 15.0).

fMRI image acquisition

Imaging was performed in a 3 Tesla GE Signa scanner using a standard 16 rung bird-cage
head coil. The head was packed with foam pads and strapped with a headband to avoid
head movement. In each run 96 whole T2*-weighed spiral-out brain scans were acquired
(24 axial slices, 5 mm thick with no gap). The field of view was 24 cm with a 64 ×
64 matrix. Repetition time (TR) was 1500 ms, echo time (TE) 24 ms and flip angle 60°.
T1-weighed structural scans for anatomical localization (60 slices, 0.95 mm thick)
were also obtained (field of view 24 cm, 256 × 256 matrix, TR 200 ms, TE 3.4 ms, flip
angle 90°). The stimulation paradigm was controlled by computer software (E-prime,
Psychology Software Tools, Pittsburg, Pennsylvania). The software also stored subjective
ratings via the 5-button claw strapped to the subject's right hand. The computer running
E-prime was triggered by the onset of the scanner radio frequency (RF) pulse. The
computer then delivered trigger signals to the stimulator. Exact stimulus onset times
were stored for later use during the statistical modeling.

Messages were presented to the subject via head mounted MRI compatible LCD display
(Resonance Technologies, Los Angeles, CA). Before each stimulus, a 'READY' screen
was displayed for 2 to 6 s. At the offset of 'READY' the stimulus was delivered. The
following 'WAIT' screen lasted 5 s before 'RATE STIMULUS' was displayed for 4 s, at
which time the subject rated the stimulus by pushing one of the five buttons (Figure
1). After each run there was a 2-3 min break. The modalities were presented in a pseudorandom
sequence between runs. To maintain the subject's alertness, they were told that over
one run the stimulus intensity could vary slightly, or could be the same, and that
a purpose of the study was to determine their ability to detect this.

Definition of volumes of interest (VOI)

Based on the previous experience in this laboratory and the results of published pain
activation studies [6,7], volumes of interest (VOI) were defined anatomically as boxes, based on the brain
anatomy atlas by Talairach and Tornoux [19]. VOI included the primary and secondary somatosensory cortex (S1/S2), anterior and
posterior insula (AIns/PIns), pregenual -, anterior mid-, posterior mid-, and dorsal
posterior cingulate cortex (pACC, aMCC, pMCC, dPCC), hippocampus (Hippo), thalamus
(Thal), hypothalamus (Hypo), and posterior orbitofrontal cortex (POFC) bilaterally
(the latter delineated within a 13-mm radius sphere). For the cingulate cortex, we
adopted the anatomical terminology used by Vogt [20]. Several structures were divided into two or more VOI (e.g. Hippocampus). Table 1 presents the min-max Talairach coordinates in each direction for the 36 VOI. Negative
x-coordinates are ipsilateral to the stimulus (left side of brain). A binary image
(mask) was generated for each VOI.

Group level analysis of main effects and contrasts between intensities

The volumes were motion corrected (MCFLIRT) and spatially smoothed (5-mm FWHM Gaussian
kernel) and high-pass filtered (50 s). General linear model analysis was carried out
using FEAT (FMRI Expert Analysis Tool) Version 5.4, part of FSL (FMRIB's Software
Library) [21]. The trials in each run were modeled as delta functions convolved with the gamma
function and its first derivative, as implemented in FSL. The first three volumes
in each run were deleted to achieve steady-state magnetization. The contrast images
were then spatially normalized to MNI-space (2.0, 2.0, 2.0 mm) and analyzed in a higher-level
mixed-effects analysis. Contrasts were defined for main effects of stimulus intensity
(41 and 51) and for differences between stimulus intensities (i.e. 41 < 51 and 41
> 51). Higher-level analysis was carried out using FLAME (FMRIB's Local Analysis of
Mixed Effects) [22,23]. Finally, for each contrast, a one-sample T-test was performed on the results of
the mixed effects model using SPM2 [24]. Significant activations were calculated at group level within each of the a priori
defined VOI. The VOI masks were used as search volume when activation maps were assigned
a statistical threshold of p < 0.01, corrected for multiple comparisons by using the
theory of random fields [25] and SPM2's small-volume correction (S.V.C.) function. Within each VOI, the number
of suprathreshold (p < 0.01, S.V.C. corrected) voxels is reported, in addition to
the Z-score and Talairach coordinates of the most significant voxel.

Time series analysis

For each VOI and stimulus intensity, single trial averages were calculated from the
individual BOLD responses. First, an average BOLD response of the significant voxels
(p < 0.01, uncorrected) was calculated across the four runs per stimulus intensity
for each subject. Second, an average BOLD response was calculated across subjects,
resulting in 72 BOLD responses (36 VOI × 2 stimulus intensities). The BOLD responses
were extracted from the functional images in subject space by inverse transformation
of the 36 VOI mask images. Voxelwise normalization over the time course was achieved
by dividing the signal intensity at each time point by the voxel's mean intensity.
All calculations were done using custom made Matlab software (Matlab 7.0, The Mathworks
Inc., Natick, MA, USA) [26]. We identified the peak latencies of each BOLD response in order to categorize VOI
that showed an Aδ-predominant response from VOI that responded to Aδ-

and

C-fiber input. A later peak latency was taken as a contribution from C-fibers. Thus,
VOI were defined as differentiating between Aδ and C fiber inputs if the peak BOLD
latency at 51°C stimulation was shorter than the peak BOLD latency at 41°C stimulation.

A major difference between the time series analysis and the mixed effects analysis
at group level is that no statistical comparison is made between the two stimulus
modalities in the time series analysis. This distinction means that a higher BOLD
amplitude following one stimulus modality compared to another does not necessarily
mean higher activation. A statistical comparison between modalities, such as the mixed
effects analysis above, is necessary to conclude that one stimulus gives more reliable
activation than another.

In summary, the identification of Aδ-predominant structures based on the functional
imaging data was accomplished by combining three criteria: i) mixed effects analysis,
identifying main effects (at 41°C and 51°C), ii) differences in the amplitude of the
responses to different stimuli (51 > 41°C and 51 < 41°C); and iii) by latency analysis
of the averaged BOLD responses (i.e. single trial averages) at 41°C and 51°C.

Results

Evoked potentials

Contact heat evoked potentials (CHEPs) were identifiable in all but one subject. The
grand average responses at Cz after ten 41°C stimuli (major positive peak latency:
554 ms) and ten 51°C stimuli (352 ms) across subjects are shown in Figure 2A and 2B, respectively. When average responses following a 41°C stimulus are quantified for
each subject before averaging, the mean latency of the major positive potential is
somewhat higher (598.0 ± 130.7 ms; Figure 3A). Similarly, the 51°C stimulus yields a mean latency (individual peaks averaged across
subjects) of 387.7 ± 96.2 ms (Figure 3A). The difference between the two main positive peaks for 41°C and 51°C was statistically
significant (p = 0.04). These latencies were similar to data presented in Granovsky
et al.[14] (599.1 ± 134 ms and 405 ± 48 ms, respectively) and the latency difference is consistent
with strongly predominant C fiber excitation by the 41°C stimulus and Aδ fiber excitation
by the 51°C stimulus. The small, ultralate positive potential shown around 660 ms
following the 51°C stimulus (Figure 2B) suggests the possibility of C fiber excitation, but this was not apparent in the
psychophysical results (see below).

Figure 2.Vertex (Cz) potentials evoked by contact-heat pulses applied to the hairy skin of
the left hand. Grand average responses were averaged across subjects after 10 stimuli delivered
at 41°C (A) and at 51°C (B). Dotted lines mark the latencies of the major positive
peaks (A and B).

Figure 3.Latency of major positive peak and pain intensity (NRS). A) Latency of the first major positivity in the heat-evoked potential at the vertex
(electrode Cz). Latency of 51°C stimuli were significantly shorter than latency of
41°C stimuli (*p = 0.04). B) Average pain intensity ratings obtained during scanning.
Ratings of 51°C stimuli were significantly more intense than ratings of 41°C stimuli
(*p = 0.018). Values are mean ± standard deviation.

Psychophysics

The average rating of the 51°C stimuli was 2.9 ± 0.8 (mean ± standard deviation; S.D.)
on the 5-point NRS (Figure 3B). This was significantly higher than the average rating of the 41°C stimuli, 2.1
± 0.5, (p = 0.018; Figure 3B). Therefore, the psychophysical results are consistent with a clear differentiation
between the sharp pain associated with brief Aδ fiber stimulation and a predominantly
warm, painless sensation associated with the brief excitation of C fiber activity.
Subjects did not report a second-pain sensation after the 51°C stimuli.

fMRI analysis

We sought to determine whether the cutaneous stimulation of Aδ fibers differentially
activates structures that are anatomically distinct from structures activated by the
cutaneous heat stimulation of C fibers.

Group results for main effects

Of the 36 structures analyzed (small volume corrected; p = 0.01 threshold; Table 1), eight structures responded to the 51°C stimulation (three bilaterally, three contralaterally
only, and two ipsilaterally only), and two structures responded exclusively to the
51°C stimulation (one ipsilateral and one contralateral), Table 2A. Eleven structures responded to the 41°C stimulation (six bilaterally, four contralaterally
only, one ipsilaterally only), and seven structures responded exclusively to the 41°C
stimulation (one bilateral, six contralateral, and four ipsilateral), Table 2B. Seven structures responded to both stimulus intensities (two bilaterally, three
contralaterally only, and two ipsilaterally only). Thus, eight structures in Table
2A are initial candidates for being predominantly Aδ-responsive structures because they
responded to the 51°C stimulus: bilateral A Ins, pACC and POFC, contralateral aMCC,
hippocampus and S2, and ipsilateral P Ins and hypothalamus. The response to 51°C (Table
2A) is a necessary, but not sufficient, criterion for a structure to be identified as
being predominantly Aδ-responsive, because activations in Table 2 are not based on a statistical comparison between stimulus intensities. To determine
whether a structure responds predominantly to Aδ fiber stimulation, it is necessary
to contrast the responses to these stimulus modalities.

Group results for contrast of stimulus responses

Table 3 shows the voxel counts and z-scores obtained from contrasting the parameter estimates
from the responses to 51°C and 41°C stimuli. This contrast shows activation in the
bilateral anterior insulae, the contralateral hippocampus, and the ipsilateral lateral
thalamus, posterior orbitofrontal cortex, posterior insula, and perigenual anterior
cingulate cortex. Two single voxel activations were in the posterior corpus callosum
and not considered further. A cluster of five voxels in the ipsilateral ventricle
is also not considered further. Conversely, if 51°C activation maps are subtracted
from 41°C activation maps, the resulting contrast reveals structures that respond
predominantly to 41°C; this includes all structures in Table 3 plus the bilateral secondary (S2) somatosensory cortex, contralateral lateral thalamus,
and hypothalamus, and the ipsilateral anterior and posterior mid-cingulate cortex,
dorsal posterior cingulate cortex, primary (S1) somatosensory cortex, hypothalamus,
and hippocampus. A detailed presentation of these activations is not given since structures
that respond predominantly to 41°C are beyond the scope of this paper. A small cluster
at the border of the ipsilateral posterior insula and the opercular-temporal lobe
junction appears also in this contrast but not in the main effects analysis. The ipsilateral
medial thalamus, anterior insula, and caudal anterior cingulate cortex, although activated
in the previous comparison, are not activated in this one.

Time series analysis

A complimentary analysis in searching for Aδ-predominant structures was to perform
a time series analysis of the BOLD responses. The latency analysis identified 11 contralateral
and 9 ipsilateral structures that satisfied the latency criterion (Table 4). The average peak latency was 8.0 ± 0.8 s after the 41°C stimulation and 6.7 ± 1.1
s after the 51°C stimulation (t = 4.9; p < 0.001).

Aδ-predominant structures

To identify structures that have a predominant response to Aδ fiber and C fiber stimulation,
we applied three criteria to our BOLD data: activation at 51°C (main effects), larger
activation at 51°C than 41°C (contrast of stimulus responses), and shorter 51°C latency
than 41°C latency (time series analysis). Table 5 lists the structures fulfilling each of the three criteria and the structures fulfilling
all three criteria: the bilateral A Ins, ipsilateral P Ins and contralateral hippocampus.
Notably, the ipsilateral P Insula fulfilled these criteria and also responded exclusively
during the 51°C stimulation. Figure 4 shows the average BOLD-response to 41°C and 51°C stimulation in the structures meeting
all three criteria. Please note that the time series analysis is normalized and does
not allow one to conclude that a higher BOLD amplitude equals stronger activation.
No statistical comparison is done between the 41°C and 51°C BOLD responses (see also
Methods). The time series analysis defines a structure as Aδ predominant or not solely
based on the peak latencies of the two BOLD responses (indicating an earlier processing
of 51°C stimuli than 41°C stimuli). Figure 5 shows brain locations of the Aδ predominant activations. In addition to the four
structures satisfying all criteria, pACC responded exclusively to 51°C by the first
criterion (Table 2A).

Figure 4.BOLD responses of Aδ predominant structures. Responses are from bilateral A Ins, contralateral hippocampus and ipsilateral P
Ins. Each trace is the average BOLD response across subjects for 41°C (open circles)
and 51°C (filled circles) ± standard error of the mean. The y-axis indicates % change
from baseline (calculated as the average BOLD response over the preceding 10 s). Time
resolution is 1.5 s (= TR). The 51°C BOLD peaks 1.5 - 3.0 s earlier than the 41°C
BOLD in all these VOI, which is one of the three criteria established for Aδ predominant
structures, as described in the text.

Figure 5.Brain locations of Aδ predominant activation. Activations in the bilateral A Ins, ipsilateral P Ins and contralateral hippocampus
satisfied three criteria for being Aδ predominant: activation at 51°C, larger activation
at 51°C than 41°C, and shorter 51°C BOLD latency than 41°C BOLD latency. Z-coordinates
are in Talairach measurements.

Discussion

Our findings show that two distinct sets of forebrain structures participate in elaborating
the perception of "first pain" that follows the preferential excitation of Aδ heat
nociceptors: 1) those preferentially responsive to noxious stimulation of Aδ fibers
and 2) those with similar responses to converging inputs from the innocuous stimulation
of C fibers. We used cerebral potentials to identify stimuli that predominantly stimulate
Aδ or C fibers. We then used these stimuli in an fMRI contrast paradigm to identify
structures that were preferentially active during the pain associated with the brief
excitation of Aδ heat nociceptors as compared with the innocuous warmth associated
with the brief excitation of heat-sensitive C fibers. Because this image contrast
reflects both heat pain and preferential Aδ excitation, the relative contribution
to the contrast of each of these variables is unknown. An experiment comparing painless
and painful selective excitation of both Aδ and C heat receptors could address this
question but would be challenged seriously by the strong association of Aδ heat receptors
with pain and the temporal summation required to evoke heat pain mediated only by
C fibers [12,14,15,27]. Meanwhile, the evidence presented here strongly favors the interpretation that the
bilateral anterior insula (A Ins), contralateral hippocampus (Hipp), and ipsilateral
posterior insula (P Ins) play a unique and determining role in mediating the acute
heat pain associated with the excitation of Aδ heat nociceptors.

Fiber specificity of the stimuli

By selecting stimulus temperatures and measuring the latencies of contact heat evoked
potentials, we assured that nociceptive Aδ fibers were stimulated preferentially by
51°C and that C fibers were stimulated preferentially by 41°C. This assessment is
based on the psychophysical measurements and the CHEP results, which were essentially
identical to data presented in Granovsky et al. [14] for hairy skin. We cannot completely rule out Aδ fiber contributions at 41°C, because
some Aδ fibers respond to temperatures near 40°C in hairy skin [28]; however, we did not detect psychophysical or evoked potential evidence for Aδ fiber
responses to this stimulus. The 51°C heat stimulation probably excited type 2, and
possibly some type 1, Aδ heat nociceptors. Although heat-sensitive C afferents must
be excited also by this stimulus, the only suggestion of a C fiber response was the
attenuated positive wave shown around 660 ms in Figure 2B. This has been observed in some laser evoked potential studies [15,29], but the fiber type, if any, associated with this response is unknown and, in our
study, there was no psychophysical characteristic associated with it. That is, the
subjects did not report on a second, delayed, heat sensation after the 51°C stimuli.
This observation is consistent with previous investigations [14,30] and the suppression of C fiber evoked responses following the excitation of Aδ fibers
[17,31]. Furthermore, the 51 > 41 fMRI contrast excludes activations mediated by the most
heat-sensitive C afferents, leaving only the possibility of some fMRI activations
by high threshold C heat nociceptors that did not evoke a cerebral potential. The
weak stimulation of heat nociceptors evokes innocuous sensations of warmth [32], so some of the innocuous heat stimuli we applied may have excited low threshold
C heat nociceptors.

Structures differentiating first from second pain

We did not elicit a clearly painful sensation with contact heat stimuli that evoked
a cerebral potential mediated by C fibers without evidence for Aδ fiber excitation.
The inability to evoke pain reliably, if at all, with single, brief C fiber selective
stimuli is in accord with previous studies [9,14,17,27,29,31,33-37]. Indeed, when psychophysical measures have been obtained, these brief C fiber stimuli
have been rated below pain threshold. It is notable that the phenomenon of "second
pain" is by definition preceded by an Aδ fiber-mediated painful stimulus, a condition
neurophysiologically quite different from a C fiber stimulus without a detectable
preceding Aδ component. Furthermore, a robust and reliable elicitation of pain during
selective stimulation of cutaneous C fibers is likely to require greater temporal
and spatial central summation than can be achieved with the single brief stimuli used
in evoked potential studies [38,39].

Because we were unable to directly compare and contrast structures active during "second
pain" alone with those activated during "first pain", it is possible that two of the
structures we have identified as responding preferentially during "first pain", the
bilateral anterior insulae and contralateral hippocampus, would also be active during
"second pain" because these structures also showed responses to the innocuous C fiber
stimulus; this does not, of course, preclude their function as critical determinants
in the mediation of "first pain". The ipsilateral P Ins, however, responded

only

during the 51°C stimulation, so the activity in this structure appears to be uniquely
associated with the experience of "first pain".

Posterior Insula

It is not surprising that the posterior insula is differentially and perhaps uniquely
responsive during what may be considered among the most intense, salient, alerting,
and well localized pain experiences. The posterior insula is connected primarily with
cortical areas related to somatosensory, auditory, and visual sensory functions [5,40]. The observations of Greenspan and colleagues [41] show that posterior insular lesions are associated with a significant increase in
pain tolerance, but not threshold, as assessed by the cold pressor test. A small number
of neurons responding to noxious stimuli have been recorded from the posterior insula
of the unanesthetized monkey [42]. Single neurons responding to both innocuous and noxious somatic stimuli were localized
in the more posterior granular area of monkeys [43]. Stimulation within the posterior insula evoked painful sensations at 17 of 93 (18%)
insular stimulation sites of 14 patients [44]. The patients described the sensations as burning, stinging, or disabling, and located
at well-defined somatic sites; these effects were more frequently elicited from the
posterior than the anterior insula where visceral sensations were evoked. The pain-related
region identified by these investigators overlaps the dorsal posterior insular region
activated by heat pain in the functional imaging study of Craig and colleagues [45]. Indeed, the mid-posterior insula is among the most regularly responsive regions
found among a variety of functional imaging studies [7,8,46].

Anterior Insula

Several studies suggest that the anterior insula is an essential component of the
cortical network mediating early aspects of pain perception including the anticipation
of pain; this is consistent with the differential, but not exclusive, response of
these structures during "first pain". The anterior insula is predominantly connected
with cortical areas related to limbic, olfactroy, gustatory, and viscero-autonomic
functions; it receives input from entorhinal cortex, and sends projections to the
entorhinal, periamygdaloid, and anterior cingulate cortices [40]. All responsive neurons in the primate anterior insula had large receptive fields
to innocuous somatic stimuli; however, the investigators searched for responses with
innocuous stimuli only [47]. In the study of Greenspan and colleagues [41], 2 patients with lesions involving the anterior insula had normal heat pain thresholds;
and 3 patients with anterior insular sparing but involvement of both S2 and posterior
insula, had elevated thresholds for heat or mechanically induced pain. In awake humans,
stimulation of the anterior insula produced visceral sensory experiences and visceral
motor responses, but not reports of pain [48]. However, Afif and colleagues have recently reported that cephalic pain and painful
pin-prick body sensations are evoked during electrical stimulation of the middle short
gyrus in the anterior, but not posterior, insula [49]. Early imaging studies revealed pain-related activity in the anterior insula [50-53], but activation during the first 10 s of repetitive noxious heat stimulation shifts
from the anterior to the posterior insula as stimulation continues for 45 s [54]. Ploghaus and colleagues showed that the anterior insula was active specifically
during the anticipation of experimentally induced pain rather than during the experience
of pain itself [55]. Porro and colleagues [56] confirmed the relationship of anterior insular activity and pain anticipation but
also found that it correlated with perceived pain intensity. The anterior insula is
also among the brain structures responding specifically to stimulus novelty and salience,
consistent with its differential activation during "first pain" [57]. Together with the results of our study, the observations cited above suggest that
anterior insular activation is related primarily to imparting salience, arousal, and
motivation to pain rather than the performance of sensory discriminative functions.

Hippocampus

The entorhinal cortex, the main input pathway to the hippocampus, has strong reciprocal
connections with the dysgranular and agranular cortex of the anterior insula [40], providing an anatomical substrate for the conjoint responses of the hippocampus
and anterior insulae during Aδ-mediated "first pain". Hippocampal activity has long
been associated with mnemonic and emotional functions [58,59]. The hippocampus and entorhinal cortex may be regarded as components of a fronto-temporal
cortical network for the encoding, storage, and retrieval of affectively significant
sensory information emanating in part from parietal somatosensory association areas
[60,61]. Although nociceptive responses of single hippocampal neurons have been reported
in the anesthetized rodent [62,63], comparable studies have not been performed in monkeys or humans. Hippocampal activation
has been observed in functional imaging studies of pain, especially in designs that
detect responses related to unpredictability, anxiety, or fear [58,64,65]. Given the abrupt onset, intensity, and arousing capacity of Aδ-mediated "first pain",
the differential response of the hippocampus is not surprising.

Comparison with other functional imaging studies

Qiu and colleagues [12] used very brief (1 ms) infrared laser stimulation presumed to differentially stimulate
Aδ and C fibers and report a pattern of overlapping activations that differs from
ours (bilateral thalamus, bilateral S2 cortex, bilateral ACC, and ipsilateral mid-insula).
Most notably, Qiu and colleagues did not detect any structure in which the selective
stimulation of Aδ afferents evoked a unique or greater response than the selective
stimulation of C fibers. There are several differences between our experiment and
those of Qiu and colleagues that could explain the discrepancy: 1) we contrasted the
responses evoked by painful 51°C and painless 41°C stimulation to identify activations
related specifically to Aδ-mediated heat pain; 2) we used contact heat (51°C) stimulation
of hairy skin, rather than an infrared laser, to stimulate Aδ fibers preferentially
and; 3) verified the preferential characteristic of our stimulation by evoked cerebral
potential recording and in-scanner psychophysical intensity ratings. In a related
experiment, Veldhuijzen and colleagues used a diode laser to investigate the different
forebrain responses to pricking and burning pain evoked by short (60 ms) and long
(2 s) focused (1 mm diameter) stimuli presumably selective for Aδ and C fibers, respectively
[13]. In that study, the different pain sensations were of equal intensity and unpleasantness
and there was considerable activation overlap; nonetheless, a group contrast analysis
revealed stronger activations during pricking pain bilaterally in the parahippocampal
and fusiform gyri, in the ipsilateral hippocampus, and contralaterally in the cerebellum
and occipito-parietal cuneus region. Only the ipsilateral dorsolateral prefrontal
cortex showed stronger activation during burning pain. These contrasts reflect differences
in forebrain processes mediating two equally intense and unpleasant pain conditions
and not the activation due to pain; some of these activation differences are likely
related to the excitation of different nociceptive afferents. Our experiment, however,
reveals the forebrain response associated with the pain of preferential Aδ heat nociceptor
excitation as compared to the painless warmth during the preferential excitation of
heat-sensitive C fibers.

Conclusions

These findings show that two sets of forebrain structures mediate the initial sharp
pain evoked by brief cutaneous heat stimulation: those responding preferentially to
the brief stimulation of Aδ heat nociceptors and those with similar responses to converging
inputs from the painless stimulation of C fibers. Our results suggest a unique and
specific physiological basis, at the forebrain level, for the "first pain" sensation
that has long been attributed to Aδ fiber stimulation and support the concept that
both specific and convergent mechanisms act concurrently to mediate pain.

Competing interests

The authors declare that they have no competing interests.

Authors' contributions

DAM carried out the data acquisition (evoked potentials and fMRI) and drafted the
manuscript. LHG contributed significantly to software programming, study design and
statistical analysis of the fMRI data. TDT participated in the design and analysis
of the evoked potential data. KLC participated in the design of the study, in data
analysis and contributed significantly to the manuscript. All authors read and approved
the final manuscript.

Acknowledgements

Heng Wang is gratefully acknowledged for his technical support through software programming.

This research was supported by NIAMS AR46045 and the Dept. of Veteran's Affairs. (KLC);
The Research Council of Norway (DM); John J. Bonica (IASP), IBRO, and INS Fellowship
(NIH/WHO) F05 NS 048581-01 (TDT).